Antigenic preparations

ABSTRACT

The present invention relates to antigenic preparations comprising polysaccharides and/or glycopeptides preparable from keratinophilic fungi as well as yeasts, process for the preparation of these antigenic preparations, their use as pharmaceutical substances as well as their use as vaccines, including but not limited to, the prophylaxis and treatment of allergy, as well as for modulating the immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/011,018,filed on May 15, 1998, allowed, which is a 371 of PCT/EP96/03535, filedon Aug. 9, 1996, which was published under PCT Article 21(2) in Englishon Feb. 27, 1997, the contents of each of which are entirelyincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antigenic preparations comprisingpolysaccharides and/or glycopeptides preparable from keratinophilicfungi as well as yeasts, processes for the preparation of theseantigenic preparations, their use as pharmaceutical substances as wellas their use as vaccines, including but not limited to, the prophylaxisand treatment of allergy, as well as for modulating the immune response.

2. Description of Related Art

Allergy in one form or another afflicts more than 20 per cent of thehuman population, and the alarming increase in its prevalence, morbidityand mortality over the past decade has led to its designation as thenumber one environmental disease (Sutton and Gould, Nature 1993, 366,pp. 421–428). Human and animal populations are afflicted by allergy to asimilar extent.

In the development of allergy, immunological reactions play a key role(Paul, William E.(Editor), Fundamental Immunology, Raven Press Books,Ltd., New York, 1984). In principle two different types of allergicreactions have been described. One is immediate type hypersensitivity(ITH), for which the maximum allergic response to the allergen isobserved within minutes to hours. The second is delayed typehypersensitivity (DTH). In case of DTH, the allergic response to theallergen usually reaches its maximum after 24 to 48 hours. Most likelyITH is mediated predominantly via the IgE pathway, whereas DTH is morecomplex. In the development of DTH it is likely that further cellmediated responses (i.e. B- and T-lymphocytes) are involved. Forexample, after transferring lymphocytes and antibodies from allergicdonor animals to non-allergic recipient animals, the recipientsdeveloped DTH (Askenase, P. W. (1973), J. exp. Med., 138, pp.1144–1155).

Because of their direct exposure to environmental antigens, tissues mostafflicted by allergies are the epithelial tissues, especially the skin.For example, in the dermatological clinic, acute allergic contactdermatitis and chronic allergic contact eczema account for up to 15% ofall dermatoses. Allergic asthma accounts for about 20% of all asthmacases in humans.

Allergic diseases that can be classified as ITH, are for example atopiceczema, allergic bronchial asthma, hay fever, rhinitis, conjunctivitis.These can develop into chronic forms as well and should not beconsidered exclusively as IgE-dependent reactions. Examples of DTH areacute allergic contact dermatitis and chronic allergic contact eczema,which can further be classified as DTH (type IV) with epidermalinvolvement. Such a patient would have previously been sensitisedthrough contact with an allergen and has developed hypersensitivity.Renewed contact with the allergen results in acute, sub-acute or chronicinflammatory contact dermatitis.

One example for an allergic dermatitis from the veterinary clinic isSummer Eczema, also called Sweet or Queens land Itch. Summer Eczema isan allergic dermatitis of horses, belonging to the atopic form ofallergic diseases (involving Type I and IV reactions). Summer Eczema isprovoked by the bite of midges of the families Culicidae andCeratopgonidae, and characterised by skin lesions with permanenterosions and exudations, mainly in regions of the mane, tail, andabdomen. Afflicted animals display a strong sensitivity of the skin withregard to irritations, i.e. touch, rain, wind etc., impairing theiroverall health and performance. As with other allergies, it is believedthat the development of this disease is also influenced by nutritionalfactors. The symptoms of this disease are only visible from March toSeptember, whereas the allergen induced sensitivity of the skin isobserved during the whole year. Summer Eczema provides aninteresting-general model system for the study of allergy and for thedevelopment of anti-allergic substances.

Many treatments for allergy have been proposed, depending on theclinical picture. For the treatment of acute allergic contactdermatitis, chronic allergic contact eczema and/or atopic eczema usuallylipophilic creams comprising glucocorticosteroids, anti-microbialsubstances, anti-inflammatory drugs and/or calcium are used. For thetreatment of Summer Eczema various compounds have been applied locallyor parenterally, for example steroid preparations, insecticides,different galenic formulations, salicylates, oils or peptides isolatedfrom micro-organisms. All of the above treatments only deal with thesymptoms and not the causes of allergy.

Impaired immune response or immunodeficiency often play important rolesin the development of allergy. Therefore, also immunotherapeuticmethods, for example the administration of immune-stimulators like BCG,levamisol and other stimulators, have been used for the treatment ofeczema, atopic eczema, skin abscesses, and also auto-immune diseases (A.M. Tschernucha (Editor), Koscha, published by Medicina in 1982, Moscow).

For the treatment of flea-allergic dermatitis, the administration ofantibody derived peptides has been successfully used (British patentapplication No 8913737). For the treatment of atopic eczema,desensitivisation has also been used with relatively good results (A. M.Tschernucha (Editor), Koscha, published by Medicina in 1982, Moscow).

In spite of the various different approaches in treating allergy, to ourknowledge, no antigenic compounds preparable from keratinophilic fungior yeasts have been used for the treatment of allergy.

In the context of the present invention the term “soluble” or“nonsoluble” refers to the solubility in aqueous solution. The term“antigenic preparation” refers to any composition of matter that is ableto elicit an antigenic or immunogenic response. The term “modulating theimmune response” refers to the ability of the antigenic preparations ofthe present invention to stimulate or enhance the immune response, forexample as demonstrated by their ability to stimulate the proliferationof lymphocytes in cell culture, (a detailed review can be found inStrube et al. (1989) Vet. Med. Rev., 60, pp. 3–15, Büttner M. (1993)Comp. Immun. Microbiol. Infect. Dis., 16, No. 1, pp. 1–10).

BRIEF SUMMARY OF THE INVENTION

It has now been surprisingly found, that antigenic preparationspreparable from keratinophilic fungi or yeast can be used for theprophylaxis and treatment of allergies, as well as for modulating theimmune response, particularly in mammals.

DETAILED DESCRIPTION OF THE INVENTION

Processes for preparing antigenic material from keratinophilic fungi aswell as yeasts have now been developed. The antigenic preparationspreparable according to these processes comprise polysaccharides and/orglycopeptides. The antigenic preparations can be used as pharmaceuticalcompositions as well as vaccines for the treatment of animals andhumans, especially for the treatment of allergies and for modulating theimmune response. It will be understood that the pharmaceuticalcompositions of this invention can have immunological as well aspharmacological utility.

The antigenic material of this invention may also be prepared frommaterial derived from keratinophilic fungi or yeasts, for example fromthe fungal or yeast cell walls.

For the preparation of the antigenic preparations of the presentinvention, three different processes have been developed. According tothese processes three different antigenic fractions (ASMP, ANMP orAEMP), in the following commonly referred to as “fractions”, can beprepared from keratinophilic fungi as well as yeasts. Antigenicpreparations comprising more than one fraction are referred to in thefollowing as “complex preparation” or abbreviated “Complex”.

Process 1: The fraction preparable according to this process consists ofantigenic soluble material comprising polysaccharide and/orglycopeptides (ASMP).

Briefly this process, which is illustrated in detail in Example 1,comprises the following:

Keratinophilic fungi or yeasts are cultivated on Agar plates, forexample as described in EP 0564620. One preferred medium is for examplemalt extract agar from Oxoid. Other media that will ensure growth ofkeratinophilic fungi or yeast may be used as well. The resulting fungalbiomass is lifted off and treated with an aqueous solution of alkali.Preferred aqueous alkaline solutions are NaOH or KOH at preferredconcentrations of 0.1–5% (w/v). Alkaline treatment is preferably at20–150 C for up to 30 h. Following the processing under aqueous alkalineconditions, the solid and liquid phases of the preparation areseparated, for is example by centrifugation, filtration orsedimentation. Preferably the separation is achieved by centrifugation,which ensures good separation of the fungal cell debris, for example atforces of about 3500 g. The treatment under aqueous alkaline conditions,as well as the separation step, may be repeated several times.

After the alkaline treatment, the resulting supernatant is treated underacidic aqueous conditions, e.g. 0.2–1.5M organic acid or 0.05–1M mineralacid. For example HCl or acetic acid can be used, preferably at pHvalues between pH 2.5 and pH 4.5. Preferably the treatment under aqueousacidic conditions is for 2 to 4 hours at temperatures of 4 to 8 C,whereafter separation of the solid and liquid layers takes place. Thetreatment under aqueous acidic conditions, as well as the separationstep, may be repeated several times, preferably under conditions asabove indicated. Then, the supernatant from the separation step issubject to a precipitation step. Preferably the precipitation isperformed by adding a suitable organic solvent, e.g. an alcohol such asa lower alkanol to the supernatant, for example methanol or ethanol. Aratio of one volume supernatant to 2–5 volumes of alcohol will result ingood-precipitation of the antigenic material. Other non-alcoholicprecipitation procedures known to the person skilled in the art may beused as well, for example ammonium sulphate or other salt precipitationmay result in precipitation of the antigenic material as well. The solidphase is then subject to a further separation step, preferably underconditions as described above. The resulting solid phase is recoveredand if desired is dissolved in an aqueous solution, preferably indistilled water, typically 25 to 100 ml are used. Finally the ASMPpreparation can be lyophilised and stored for prolonged time periodsunder dry conditions.

Process 2: The fraction preparable according to this process consists ofantigenic nonsoluble material comprising polysaccharide and/orglycopeptides (ANMP). Briefly this process, which is illustrated indetail in Example 2, comprises the following:

Keratinophilic fungi or yeasts are cultivated on Agar plates, forexample as described in EP 0564620. A preferred medium is for examplemalt extract agar from Oxoid. Other media that will ensure growth ofkeratinophilic fungi or yeast may be used as well. The resulting fungalbiomass is lifted off and treated with an aqueous solution of alkali.Preferred aqueous alkaline solutions are NaOH or KOH at preferredconcentrations of 0.1–5% (w/v). Alkaline treatment is preferably at20–150 C for up to 30 h. Following the processing under aqueous alkalineconditions, the solid and liquid phases of the preparation areseparated, for example, by centrifugation, filtration or sedimentation.Preferably the separation is achieved by centrifugation, which ensuresgood separation of the fungal cell debris, for example at forces ofabout 3500 g. The treatment under aqueous alkaline conditions may berepeated several times, as well as the separation step. After alkalinetreatment, the solid phase is treated with mineral or organic acids.Preferably 0.2–1.5 M acetic acid or 0.05–1 M HCl are added to the solidphase for 0.5 to 3 hours at temperatures of 70 to 100 C. After acidictreatment the solid phase is washed with an aqueous solution, preferablydistilled water. Advantageously the washing is repeated about fivetimes. Finally the solid phase is suspended in distilled water.

Process 3: The fraction preparable according to this process consists ofantigenic exogenous material comprising polysaccharide and/orglycopeptides (AEMP). Briefly this process, which is illustrated indetail in Example 3, comprises the following:

Keratinophilic fungi or yeasts are incubated in aqueous solution orcultivated in liquid medium for up to 240 hours (the volume of thesolution or culture is here defined as primary volume PV). Distilledwater can be used (see example 3. I.) as well as media described in EP0564620. After incubation or cultivation, the fungal cells areseparated, for example, by centrifugation, filtration or sedimentation,preferably by centrifugation under conditions as described above. Theresulting supernatant is then lyophilised and subsequently dissolved inwater. Preferably the volume of water equals 0.1 to 0.2 volumes of theprimary volume (PV). The resulting solution is then subject to aprecipitation step. Preferably the precipitation is performed by addinga suitable organic solvent, e.g. an alcohol such as a lower alkanol tothe supernatant, for example methanol or ethanol. A ratio of one volumesupernatant to 2–5 volumes of alcohol will result in good precipitationof the antigenic material. Other non-alcoholic precipitation proceduresknown to the person skilled in the art may be used as well, for exampleammonium sulphate or other salt precipitation may result inprecipitation of the antigenic material as well. The resultingprecipitate is recovered and if desired is dissolved in an aqueoussolvent, preferably in distilled water. Preferably 0.5 to 50 mg of theprecipitate are dissolved in 1 ml of aqueous solvent. Finally the AEMPsolution can be lyophilised and stored for prolonged time periods underdry conditions, preferably at 2 to 10 C.

Preferred fungal genera from which the above defined Fractions arepreparable are the genera Trichophyton, Microsporum or Candida.

Preferred species are:

-   -   Trichophyton equinum,    -   Trichophyton mentagrophytes,    -   Trichophyton sarkisovii,    -   Trichophyton verrucosum,    -   Microsporum canis,    -   Microsporum gypseum, or    -   Candida albicans.        Preferred strains of the above referenced species are:    -   Trichophyton equinum DSM No. 7276,    -   Trichophyton mentagrophytes DSM No. 7279,    -   Trichophyton sarkisovii DSM No. 7278,    -   Trichophyton verrucosum, DSM 7277,    -   Microsporum canis DSM No. 7281,    -   Microsporum canis var. obesum DSM No. 7280,    -   Microsporum canis var. distortum DSM No. 7275,    -   Microsporum gypseum DSM No. 7274, or    -   Candida albicans, DSM No. 9656.

All above referenced strains have been deposited by the applicant at theDSM (“Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH”,Mascheroder Weg 1B, D-38124 Braunschweig, Germany) under the provisionsof the Budapest Treaty on the deposition of micro-organisms. All strainsexcept Candida albicans DSM No. 9656 have been previously described inthe USSR Patent Application No. 5006861 filed 21, Oct., 1991, andcorresponding applications i.e. the published Patent Application EP0564620, filed on 17, Oct., 1992.

Depending on the species the fractions can be obtained from, they arereferred to according to the following.

Fractions derived from:

-   (i) Trichophyton equinum, are referred to as ASMP-TE, ANMP-TE, or    AEMP-TE,-   (ii) Trichophyton mentagrophytes, are referred to as ASMP-TM,    ANMP-TM, or AEMP-TM,-   (iii) Trichophyton sarkisovii, are referred to as ASMP-TS, ANMP-TS,    or AEMP-TS,-   (iv) Trichophyton verrucosum, are referred to as ASMP-TV, ANMP-TV,    or AEMP-TV,-   (v) Microsporum canis, are referred to as ASMP-MC, ANMP-MC, or    AEMP-MC,-   (vi) Microsporum gypseum, are referred to as ASMP-MG, ANMP-MG, or    AEMP-MG, or-   (vii) Candida albicans, are referred to as ASMP-CA, ANMP-CA, or    AEMP-CA.

Where information with regard to the specific stgrain is given, thespecies abbreviation is followed by the digits of the specific DSMdeposit, for example—AEMP-CA9656 refers to the AEMP fraction preparablefrom Candida albicans strain DSM No. 9656.

The Fractions preparable as defined in any one of the above describedprocesses (1 to 3) comprise at least one single antigen preparable fromat least one of the above referenced fungi. The antigenic preparationsof the present invention comprise at least one of the above definedfractions or combinations thereof.

The antigenic preparations (ASMP and AEMP) as described in Examples 1and 3:

1) comprise monosaccharides, amino acids and nucleotides, which arebound to a large extend in polymeric structures and to a smaller portionare free monomers.

2) mainly consist of the monosaccharide units: mannose galactose,glucose and xylose and others in different relative amount.

3) contain a mixture of polymeric structures formed by a significantamount of these monosaccharides. A significant part of these polymericstructures show molecular weights greater than 20 000 kD.

4) contain low amounts of free or bound amino acids.

5) contain low amounts of DNA molecules shown to be sensitive todigestion with DNase I.

NMR spectroscopy of the antigenic preparations ASMP and AEMP resulted inthe NMR spectrograms presented in FIGS. 1 to 4.

The chemical shifts and signal multiplicities (summarized in Table 12)are in agreement with literature data for carbohydrates and amino acids.

For AEMP and ASMP fractions, e.g. MG 7274, TM 7279 and CA 9656, thecarbohydrate signals cover a range from 3.2–5.5 ppm, the amino acidsignals a region from 0.75–3.45 (without α-protons).

ASMP also shows typical signals for acetate-CH₃ 1.92 ppm.

The AEMP fractions show also typical signals for disacharides and aminoacids. E.g. the TM 7279 spectrum shows signals for aromatic amino acidslike Phenylanalanine, Tyrosine and Tryptophane in the region 7.15–7.9ppm.

Concerning single fractions of ASMP or AEMP, concentrations of 0.1 to 50mg/ml are preferred. Concerning single Fractions of ANMP, concentrationsof 0.1 to 5% (v/v) are preferred.

Preferred embodiments of the antigenic preparations of the presentinvention comprise for example the following combinations of Fractions(Complexes):

Complex 1 comprises ASMP-TM, and ASMP-MG, and ASMP-CA. Preferably theconcentration of each fraction is 0.1 to 50 mg/ml. A highly preferredembodiment according to Complex 1 is a combination of ASMP-TM7279,ASMP-MG7274, and ASMP-CA9656.

Complex 1.1 comprises ASMP-MG and ASMP-CA. Preferably the concentrationof each fraction is 0.1 to 50 mg/ml. A highly preferred embodimentaccording to Complex 1.1 is a combination of ASMP-MG7274 andASMP-CA9656.

Complex 2 comprises ANMP-TM, and ANMP-MG, and ANMP-CA. Preferably theconcentration of each fraction is 0.1 to 5% (v/v). A highly preferredembodiment according to Complex 2 is a combination of ANMP-TM7279,ANMP-MG7274, and ANMP-CA9656.

Complex 3, comprises AEMP-TM, and AEMP-MG, and AEMP-CA. Preferably theconcentration of each fraction is 0.1 to 50 mg/ml. A highly preferredembodiment according to Complex 3 is a combination of AEMP-TM7279,AEMP-MG7274, and AEMP-CA9656.

Complex 4 comprises ANMP and AEMP. The following combinations offractions are preferred: (1) ANMP-CA and AEMP-TM or (2) ANMP-MG, ANMP-TMand AEMP-TM. Preferably the concentration of ANMP is 0.1 to 5% (v/v) andthat of AEMP is 0.1 to 50 mg/ml. Highly preferred embodiments accordingto Complex 4 are the following combinations:

4.1 ANMP-CA9656, and 4.2 ANMP-MG7274, and AEMP-TM7279; ANMP-TM7279, andAEMP-TM7279;

Complex 5, comprises ANMP and ASMP. A preferred combination is ANMP-MG,and ANMP-TM, and ASMP-CA. Preferably the concentration of the individualANMP fractions is 0.1 to 5% (v/v), and that of individual ASMP fractionsis 0.1 to 50 mg/ml. Highly preferred is a combination of ANMP-MG7274,and ANMP-TM7279, and ASMP-CA9656.

Further preferred antigenic complexes according to the present inventioncomprise for example: ASMP and AEMP or ASMP and AEMP and ANMP atconcentrations for ASMP and AEMP of 0.1–50 mg/ml and for ANMP atconcentrations of 0.1 to 5% (v/v).

The antigenic preparations of the present invention can be appliedtogether with suitable physiologically acceptable carriers that do notcause adverse physiological side effects, and include buffers, solutionsor adjuvants, for example salt solutions, Lactate solutions or RingerSolution. Preferred carriers are for example: Carrier A: aqueoussolution comprising 0.85% (w/v) NaCl; Carrier B: aqueous solutioncomprising 5% (w/v) Glucose, 0.3% (w/v) meat extract “lab-lemco”(Oxoid), and 0.1% (w/v) yeast extract (Oxoid); Carrier C: Medium RPMI1640 (purchased from Serva, catalogue no 12-702).

The antigenic preparations of the present invention can be applied perse or as solutions for injection, creams, sprays, aerosols, tablets andin other application forms known to the person skilled in the art. Theantigenic preparations of the present invention may further providehighly efficient vaccines.

The antigenic preparations of the present invention are able tostimulate the proliferation of cells of the immune system and therebyare able to modulate the immune response. The antigenic preparations ofthe present invention are further able to inhibit the proliferation ofhuman keratinocytes.

The antigenic preparations of the present invention may confer a highdegree of resistance against allergic reactions, particularly ofepithelial tissues, more particularly of the skin. They are of interestfor preventing and curing allergy, and in our hands have not shownadverse side effects as demonstrated in vivo in laboratory animals (i.e.guinea pigs and white mice) and horses (i.e. cross-breed and Icelandichorses).

In particular, acute allergic dermatitis and skin lesions may beeffectively cured without side effects by administering the antigenicpreparation of the present invention, i.e. by vaccination. After intramuscular is injection(s) of the antigenic preparations of the presentinvention, the symptoms of allergic inflammation of the skin, itch andthe sensitivity of the skin of individuals afflicted with allergicdermatitis may be abolished. Complete recovery from all allergicsymptoms has been achieved within 2 to 8 weeks after the final injectionand the allergen induced sensitivity of the skin to irritants wasabolished. Further, within 1 to 6 weeks after the final injection itchmay be abolished.

In a preferred embodiment, the antigenic preparations of the presentinvention provide a protection and cure for the so called Summer Eczemaof horses, especially of Icelandic horses. After 1 to 3 intra muscularor intra dermal injection(s) of the antigenic preparations of thepresent invention, horses afflicted with Summer Eczema may be cured ofor protected against Summer Eczema, preferred are complexes 1 and 1.1.

In a further preferred embodiment, the antigenic preparations of thepresent invention provide a protection and cure against alopecia inmammals. After 1 to 3 intra muscular or intra dermal injection(s) of theantigenic preparations of the present invention, mammals afflicted withalopecia may be cured of or protected against alopecia, preferred areComplexes 1 or 1.1.

In another preferred embodiment, the antigenic preparations of thepresent invention improve the hair condition and seasonal coat change ofmammals. After 1 to 3 intramuscular or intradermal injections, coatcondition may be significantly improved and in individuals afflictedwith incomplete coat change complete change to the regular season coatmay result, preferred are Complexes 1 or 1.1.

In another preferred embodiment, the antigenic preparations of thepresent invention provide a protection and cure against eczema. After 1to 3 intra dermal or intramuscular injections) of or after topicaltreatment with the antigenic preparations of the present invention,mammals, i.e. humans, afflicted with eczema, may be cured of orprotected against eczema, preferred are fractions ASMP-MG, ASMP-CA andASMP-TM, i.e. ASMP-MG7274, ASMP-CA9656 and ASMP-TM7279 or complexes 1and 1.1.

In a further preferred embodiment, the antigenic preparations of thepresent invention provide a protection and cure against neurodermitis.After topical treatment with the antigenic preparations of the presentinvention, mammals, i.e. humans, afflicted with neurodermitis, may becured of or protected against neurodermitis, preferred are fractionsASMP-MG, ASMP-CA and ASMP-TM, i.e. ASMP-MG7274, ASMP-CA9656 andASMP-TM7279 or complexes 1 and 1.1.

The antigenic preparations of the present invention may be used to treata variety of indications such as those described in “KlinischeImmunologie”, Peter, H. H. (editor), publ. 1991 by Urban &Schwarzenberg, Munich, Germany, for example:

-   1. allergic airway diseases    -   1.1. allergic rhinitis and conjunctivitis        -   1.1.1. seasonal rhino-conjunctivitis        -   1.1.2. perennial rhinitis    -   1.2. asthma bronchiale    -   1.3. status asthmaticus    -   1.4. asthma of children        -   1.4.1. obstructive lung disease after infectious            bronchiolitis        -   1.4.2. mild episodic or mild perennial asthma bronchiale        -   1.4.3. strong perennial asthma bronchiale-   2. allergic broncho pulmonary aspergillosis-   3. food allergies    -   3.1. IgE-mediated food allergy        -   3.1.1. IgE-mediated food allergy of infants        -   3.1.2. IgE-mediated food allergy of juveniles and adults    -   3.2. IgG- and T-cell-mediated food allergies    -   3.3. Intolerance to cow's milk    -   3.4. Heiner-syndrome    -   3.5. eosinophilic gastroenteropathy    -   3.6. coeliac disease-   4. Insect bite/sting allergy-   5. urticaria in all its forms    -   5.1. contact urticaria    -   5.2. urticaria concomitant with allergic reactions    -   5.3. urticaria concomitant with intolerance to additives and        inhibitors of prostaglandin synthesis (pseudo-allergy)    -   5.4. physical urticaria        -   5.4.1. dermatographia (urticaria factitia)        -   5.4.2. cholinergic and adrenergic urticaria        -   5.4.3. cold-induced urticaria        -   5.4.4. light urticaria        -   5.4.5. pressure urticaria        -   5.4.6. other rare forms of physical urticaria    -   5.5. urticarial vasculitis    -   5.6. mastocytosis and urticaria pigmentosa    -   5.7. urticaria concomitant with infectious diseases    -   5.8. urticaria concomitant with immunothyroiditis    -   5.9. urticaria and amyloidosis-   6. angioedema    -   6.1. hereditary angloneuroticoedema (HANE)    -   6.2. acquired angioneuroticoedema-   7. atopic dermatitis, atopic eczema-   8. drug related allergy

TABLE 1 Properties and characteristics of Candida albicans DSM No. 9656Properties and characteristics of the strain DSM No. 9656 Epidemicstrain No. 008 Description of 10-day colony on 10-day colony on cultureSaboraud agar is cream Saboraud agar is cream smooth, pasty, smooth,pasty, and glistening, and glistening, with folded elevated, with asegments, the margin of central depression, the the colony is margin ofthe colony is irregular, with a regular, with a diameter of 15–18 mmdiameter of 18–22 mm Morphological spherical oval blasto- spherical ovalblasto- characteristics spores measure 3.5–5 × 5– spores measure 8 μm,pseudo hyphae are 3.5–5 × 5– 5–8 μm wide, hyphae are 8 μm, pseudo hyphaeare 2–3 μm wide. 5–8 μm wide, hyphae are Chlamydospores on rice 2–3 μmwide. agar measure 13–16 μm Chlamydospores on rice in diameter agarmeasure 13–16 μm in diameter Pathogenic 30 days after intra 30 daysafter intra characteristics peritoneal injection of peritoneal injectionof 10–100 million fungal 10–100 million fungal cells into white mice,cells into white mice, 80% of the animals 80% of the animals carriedabdominal carried abdominal granulomas, granulomas, no lethal effectsare 40% of the animals died observed

The present invention further relates to Candida albicans strain DSM No.9656, which was obtained by directed selection based on stabilisation ofcultural-morphological characteristics and attenuation of epidemicstrain No. 008, which was isolated from a man in 1990.

The biological properties of Candida albicans strain DSM No. 9656 aredescribed in Table 1.

Strain Candida albicans DSM No. 9656 further differs from the epidemicstrain in its population stability, and morphological characteristicsunder long term passaging through nutrient media and lower virulence.Following the teachings for the preparation of antigenic preparations ofthe present invention, highly effective and safe antigenic preparations,according to the present invention, can be prepared from this strain.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and attain the endsand advantages mentioned as well as those inherent therein. Thecompounds, procedures and techniques described herein are presentlyrepresentative of preferred embodiments, are intended to be exemplary,and are not intended as limitations on the scope.

Having now generally described the present invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention.

EXAMPLES

For all examples the centrifugation was performed at forces between 3000g to 3500 g for about 30–50 min. The media were purchased from Oxoid(Unipath GmbH, Am Lippeglacis 6–8, 46483 Wesel, Germany) or Serva (ServaFeinbiochemica GmbH & Co. KG, Carl-Benz-Str. 7, 69115 Heidelberg,Germany). If not indicated otherwise, the fungi were cultivated asdescribed in the Oxoid catalogue “5. aktualisierte deutsche Ausgabe” orin EP 0564 620. Fungus strains used for the preparation of the antigenicpreparations of the present invention were obtained by selection andattenuation of fungus strains as described in N. V. Mazkevitch, 1981,“Spontannaja ismentchivost i kariologia nesovershennich gibov”,published by Isdatelstwo Nauka, Moscow; and Ivanova, L. G., 1992,“Sistematika, morphologitcheskaja. charakteristika, biologitcheskiisvojstva vosbuditelej dermatophitosov, obshih dlja givotnih itcheloveka”, Moscow, Library of the University of Moscow. Basicculturing techniques for mammalian cell cultures can be readily found inDoyle, Griffiths, and Newell (Eds.), Cell & Tissue Culture: LaboratoryProcedures, John Wiley & Sons (1995). For the keratinocyte assays HaCaTcells were used (Boukamp et al. (1988), J. Cell Biol., 106, pp. 761–771,and Ryle et al. (1989), Differentiation, 40, pp. 42–54) isolatedkeratinocytes or other keratinocyte cell lines can be used as well.Horse lymphocytes were isolated and cultivated as described in Friemel,H., “Immunologische Arbeitsmethoden”, published by VEB Gustav FischerVerlag, Jena, 1984; or Paul, E., “Fundamental Immunology”, published byRaven Press, New York, 1984. Radio assays were essentially performed asdescribed in Boehncke et al., 1994, Scand. J. Immunol. 39, pp. 327–332and references cited therein. NaOH, KOH, HCl and acetic acid wereprepared as aqueous solutions. If not indicated otherwise, the termsoluble refers to the solubility in aqueous solution. Physiologicallyacceptable carriers used in the experiments described below are inexample: Carrier A: aqueous solution comprising 0.85% (w/v) NaCl;Carrier B: aqueous solution comprising 5% (w/v) Glucose, 0.3% (w/v) meatextract “lab-lemco” (Oxoid), and 0.1% (w/v) yeast extract (Oxoid);Carrier C: Medium RPMI 1640 (Serva).

Example 1

Antigenic soluble material comprising polysaccharide and/orglycopeptides (ASMP) was prepared from:

Trichophyton mentagrophytes (ASMP-TM), Microsporum gypseum (ASMP-MG) orCandida albicans (ASMP-CA), according to the following procedures:

Fungi were cultivated on Agar plates as described in EP 0564620. Thefungal biomass was lifted off and for the production of:

I. ASMP-TM:

-   (i) Trichophyton mentagrophytes biomass was treated with 4.5% (w/v)    of NaOH at about 140 C for 1 hour followed by centrifugation for 45    minutes. To the supernatant a 4M solution of acetic acid was added    until a final pH of 3.5 was reached. After 2 hours the sediment was    separated by centrifugation and 3 volumes of ethanol were added to 1    volume of supernatant. The sediment resulting from the alcoholic    precipitation was sedimented by centrifugation and dissolved in    distilled water. Finally the individual ASMP preparations were    lyophilised.-   (ii) Trichophyton mentagrophytes biomass was treated with 0.2% (w/v)    of KOH at about 140° C. for 1 hour followed by centrifugation. The    supernatant was treated with a 1M solution of HCl at a final pH of    3.5 for 4 hours at 4–10 C. The sediment was then separated by    centrifugation and 2 volumes of ethanol were added to 1 volume of    supernatant. The sediment resulting from the alcoholic precipitation    was sedimented by centrifugation and dissolved in distilled water.    Finally the individual ASMP preparations were lyophilised.    II. ASMP-MG,-   (i) Microsporum gypseum biomass was treated with 0.2% (w/v) of NaOH    at about 140 C for 2 hours followed by centrifugation. The sediment    was again treated with 0.2% (w/v) of NaOH at about 140 C for 2 hours    followed by centrifugation and the procedure was repeated for a    third time. The final supernatant was then treated with a 8M    solution of acetic acid at a final pH of 3.5 for 3 hours at 18–20 C.    The sediment was then separated by centrifugation, and 3 volumes of    ethanol were added to 1 volume of supernatant. The sediment    resulting from the alcoholic precipitation was sedimented by    centrifugation and dissolved in distilled water. Finally the    individual ASMP preparations were lyophilised.-   (ii) Microsporum gypseum biomass was treated either with 3% (w/v) of    KOH at about 75 C for 6 h followed by centrifugation. The sediment    was again treated with 3% (w/v) of NaOH at about 75 C for 6 h    followed by centrifugation. The final supernatant was then treated    with a 0.5M solution of HCl at a final pH of 3.5 for 4 hours at    4–10 C. The sediment was then separated by centrifugation, and 3    volumes of methanol were added to 1 volume of supernatant. The    sediment resulting from the alcoholic precipitation was sedimented    by centrifugation and dissolved in distilled water. Finally the    individual ASMP-preparations were lyophilised.    III. ASMP-CA:-   (i) Candida albicans biomass was treated with 3.0% (w/v) of NaOH at    about 75 C for 6 h followed by centrifugation. The sediment was    again treated with 3.0% (w/v) of NaOH at about 75 C for 6 h followed    by centrifugation. The final supernatant was then treated with a 12M    solution of acetic acid at a final pH of 3.5 for 2 hours at 4–10 C.    The sediment was then separated by centrifugation, and 2 volumes of    methanol were added to 1 volume of supernatant. The sediment    resulting from the alcoholic precipitation was sedimented by    centrifugation and dissolved in distilled water. Finally the    individual ASMP preparations were lyophilised.-   (ii) Candida albicans biomass was treated with 4.5% (w/v) of KOH at    about 35 C for 3 h followed by centrifugation. The sediment was    again treated with 4.5% (w/v) of NaOH at about 35 C for 3 h followed    by centrifugation, and the procedure was repeated for a third time.    The final supernatant was then treated with a 0.25M solution of HCl    at a final pH of 3.5 for 4 hours 18–20 C. The sediment was then    separated by centrifugation, and 2 volumes of ethanol were added to    1 volume of supernatant. The sediment resulting from the alcoholic    precipitation was sedimented by centrifugation and dissolved in    distilled water. Finally the individual ASMP, preparations were    lyophilised.

Example 2

Antigenic nonsoluble material comprising polysaccharide and/orglycopeptides (ANMP) was prepared from: Trichophyton mentagrophytes(ANMP-TM), Microsporum gypseum (ANMP-MG) or Candida albicans (ANMP-CA)according to the following procedures:

Fungi were cultivated on Agar plates as described in EP 0564620. Thefungal biomass was lifted off and for the production of:

I. ANMP-TM:

-   (i) Trichophyton mentagrophytes biomass was treated with 0.2% (w/v)    NaOH at about 35 C for 24 h followed by centrifugation. The sediment    was treated with 0.3M acetic acid for about 3 hours at about 60 C    and washed five times with distilled water. Each washing step was    followed by centrifugation. The final sediment was resuspended in an    aqueous solution of 0.85% (w/v) NaCl (Carrier A) to a final    concentration of 0.5% (v/v) of ANMP-TM. The ANMP-TM preparation was    stored as suspension at 2–10 C.-   (ii) Trichophyton mentagrophytes biomass was treated with 0.2% (w/v)    KOH at about 35 C for 24 h followed by centrifugation. The sediment    was treated with 0.1 M HCl for 30 minutes at 70 C and washed five    times with distilled water. Each washing step was followed by    centrifugation. The final sediment was resuspended in RPMI 1640    (Carrier C) to a final concentration of 1.5% (v/v) of ANMP-TM. The    ANMP-TM preparation was stored as suspension at 2–10 C.    II. ANMP-MG:-   (i) Microsporum gypseum biomass was treated with 3% (w/v) NaOH at    about 75 C for 6 h followed by centrifugation. The sediment was    treated again with 3% (w/v) NaOH at about 75 C for 6 h followed by    centrifugation. The resulting sediment was treated with 0.7M acetic    acid for about 4 hours at 60 C and washed five times with distilled    water. Each washing step was followed by centrifugation. The final    sediment was resuspended in an aqueous solution comprising 5% (w/v)    glucose, 0.1% (w/v) yeast extract from Oxoid, and 0.3% (w/v) meat    extract “lab lemco” from Oxoid (Carrier B) to a final concentration    of 2.5% (v/v) of ANMP-MG. The ANMP-MG preparation was stored as    suspension at 2–10 C.-   (ii) Microsporum gypseum biomass was treated with 3% (w/v) KOH at    about 35° C. for 3 h followed by centrifugation. The sediment was    treated again with 3% (w/v) KOH at about 35° C. for 3 h followed by    centrifugation, and the procedure was repeated a third time. The    resulting sediment was treated with 0.5M HCl for 30 minutes at 80 C    and washed five times with distilled water. Each washing step was    followed by centrifugation. The final sediment was resuspended in    RPMI 1640 (Carrier C) to a final concentration of 2.0% (v/v) of    ANMP-MG. The ANMP-MG preparation was stored as suspension at 2–10 C.    III. ANMP-CA:-   (i) Candida albicans biomass was treated with 4.5% (w/v) NaOH at    about 140 C for 2 hours followed by centrifugation. The sediment was    treated again with 4.5% (w/v) NaOH at about 140 C for 2 hours    followed by centrifugation, and the procedure was repeated a third    time. The resulting sediment was treated with 1M acetic acid for 1    hour at 60 C and washed five times with distilled water. Each    washing step was followed by centrifugation. The final sediment was    resuspended in an aqueous solution of 0.85% (w/v) NaCl (Carrier A)    to a final concentration of 1.5% (v/v) of ANMP-CA. The ANMP-CA    preparation was stored as suspension at 2–10 C.-   (ii) The Candida albicans biomass was treated with 4.5% (w/v) KOH at    about 140 C for 2 hours followed by centrifugation. The sediment was    treated again with 4.5% (w/v) NaOH at about 140 C for 2 hours, and    the resulting sediment was treated with 0.1 M HCl for 30 minutes at    100 C and washed five times with distilled water. Each washing step    was followed by centrifugation. The final sediment was resuspended    in RPMI 1640 (Carrier C) to a final concentration of 2.5% (v/v) of    ANMP-CA. The ANMP-CA preparation was stored as suspension at 2–10 C.

Example 3

Antigenic exogenous material comprising polysaccharide and/orglycopeptides (AEMP), was prepared from liquid cultures of: Trichophytonmentagrophytes (AEMP-TM), Microsporum gypseum (AEMP-MG) or Candidaalbicans (AEMP-CA). The liquid cultures were cultivated under conditionsessentially as described in EP 0564620. The individual AEMP preparationswere obtained according to the following procedures.

-   I. AEMP-TM: Trichophyton mentagrophytes was incubated for 240 h at    26 C in 1000 ml distilled water. Then, the culture, containing about    1.2×10⁸ cells per ml, was centrifuged. The supernatant was    lyophilised and dissolved in 100 ml of distilled water, 3 volumes of    methanol were added and the precipitate was dissolved in aqueous    solution. The supernatant was lyophilised resulting in AEMP-TM.-   II. AEMP-MG: Microsporum gypseum was cultivated for 50 h at 28 C in    200 ml of Carrier C (RPMI 1640 medium from Serva). The culture,    containing about 3×10⁷ cells per ml, was centrifuged. The    supernatant was lyophilised and dissolved in 20 ml of distilled    water, 2 volumes of methanol were added and the precipitate was    dissolved in aqueous solution. The supernatant was lyophilised    resulting in AEMP-TM.-   III. AEMP-CA: Candida albicans was cultivated for 30 h in 800 ml of    Carrier B (1% (w/v) meat extract lab-lemco from Oxoid, 0.1% (w/v)    yeast extract from Oxoid and 5% (w/v) dextrose) at 37 C. The    culture, containing about 10⁸ cells per ml, was centrifuged. The    supernatant was lyophilised and dissolved in a small amount of    distilled water, 2 volumes of methanol were added and the    precipitate was dissolved in aqueous solution. The supernatant was    lyophilised resulting in AEMP-TM.

Example 4

The influence of different antigenic preparations on the growth ofkeratinocyte cell cultures (HaCaT cell cultures) was determined.

-   I. Antigenic fractions ASMP-TM, ANMP-TM, and AEMP-TM prepared from    Trichophyton mentagrophytes DSM No. 7279, ASMP-MG, ANMP-MG, and    AEMP-MG prepared from Microsporum gypseum DSM No. 7274, and ASMP-CA,    ANMP-CA, and AEMP-CA prepared from Candida albicans DSM No. 9656    were used in different concentrations. The ANMP fractions as    prepared according to Example 2 were lyophilised and resuspended in    PBS (Phosphate Buffered Saline with a phosphate concentration of 6.7    mM at physiological pH of about 7.2; purchased from Serva, Catalogue    No 17-516).

For cultivation 12 well tissue culture plates from Falcon (flat bottom,surface area 9.6 cm²) were used. To each well, 0.15 ml keratinocyte cellsuspension (HaCaT cells) of about 1 million cells per ml nutrient medium(RPMI 1640 supplemented with 10% (w/v) foetal calf serum), 2 ml ofnutrient medium, and 0.02–0.1 ml antigenic fraction dissolved in PBSwere added. To control wells no antigenic fraction material was added.Cultivation was performed in an incubator with 5% (v/v) CO₂ at atemperature of 37 C for about 48 hours until a confluent cell mono layerhad developed in the control wells.

Inhibition of cell growth was determined by comparing the area size ofcell sheets treated with the antigenic fractions compared to controlsnot treated with antigenic fractions (control=100%). The results areshown in Tables 2 and 3.

Inhibition of cell growth was observed at an ASMP-MG concentration of0.1 mg/ml, an ASMP-TM concentration of 0.3 mg/ml, and an ASMP-CAconcentration of 1 mg/ml. For ANMP (MG, TM, and CA) inhibition wasobserved at a concentration of 1 mg/ml. For AEMP-MG, inhibition of cellgrowth was observed at a concentration of 0.3 mg/ml, and for AEMP-TM andAEMP-CA at a concentration of 1 mg/ml.

Example 5

The influence of different antigenic fractions on the cell proliferationof horse lymphocytes was determined.

Antigenic fractions ASMP and AEMP of the fungal strains T.mentagrophytes DSM No. 7279, M. gypseum DSM No. 7274; and C. albicansDSM No. 9656 were used. A suspension of about 40 000 lymphocytes (fromIcelandic horses) per ml of nutrient medium was prepared. Nutrientmedium RPMI 1640 was supplemented with 10% (w/v) foetal calf serum.Cultivation of the lymphocytes was performed in 96 well U-bottom tissueculture plates (Falcon No 3077). 200 μl of cell suspension wasdistributed to each well and 20 μl of antigenic fraction dissolved inPBS was added. Controls were performed without addition of antigenicfraction material.

The tissue culture plates were incubated at 37 C with 5% (v/v) CO₂ for72 hours. Then the nutrient medium was changed and aH³-Thymidine-containing solution (1 μl per well) was added. A secondcultivation step for 12 hours was performed, the culture was washed withPBS. Cell proliferation was determined by radio assay techniques asdescribed in Boehncke et al., 1994, Scand. J. Immunol. 39, pp. 327–332.Measurement of the cell proliferation was performed by comparing thetest cultures with the controls not exposed to antigenic fractionmaterial. The control values were defined as 100%. The result is shownin Table 4. The individual antigenic fractions either had an inhibitingor stimulating effect on lymphocyte cell proliferation.

Example 6

This example illustrates typical complex preparations. The complexes (1to 5) described in this example have been prepared from Trichophytonmentagrophytes DSM No. 7279, Microsporum gypseum DSM No. 7274 or Candidaalbicans DSM No. 9656.

-   I. Complex 1 comprises ASMP-TM, ASMP-MG, and ASMP-CA in a suitable    carrier, in example

Concentration [mg/ml] Complex 1 A B C ASMP-TM7279 5 10 30 ASMP-MG7274 510 30 ASMP{overscore (-)}CA9656 5 10 30 in carrier in carrier in carrierA or B or C A or B or C A or B or C

-   Complex 1.1 comprises ASMP-MG, and ASMP-CA in a suitable carrier, in    example

Concentration [mg/ml] Complex 1 A B C ASMP-MG7274 5 10 30 ASMP-CA9656 510 30 in carrier in carrier in carrier A or B or C A or B or C A or B orC

-   II. Complex 2 comprises ANMP-TM, ANMP-MG, and ANMP-CA in a suitable    carrier, in example

Concentration [% (v/v)] Complex 2 A B C D ANMP-TM7279 0.5 1.0 1.5 2.5ANMP-MG7274 0.5 1.0 1.5 2.5 ANMP-CA9656 0.5 1.0 1.5 2.5 suspensionsuspension suspension suspension in carrier in carrier in carrier incarrier A or B or A or B or A or B or A or B or C C C C

-   III. Complex 3 comprises AEMP-TM, AEMP-MG, and AEMP-CA in a suitable    carrier, in example

Concentration [mg/ml] Complex 3 A B C AEMP-TM7279 5 10 30 AEMP{overscore(-)}MG7274 5 10 30 AEMP-CA9656 5 10 30 in carrier in carrier in carrierA or B or C A or B or C A or B or C

-   IV. Complex 4 comprises ANMP and AEMP in a suitable carrier, in    example-   (i) Complex 4.1 ANMP-CA9656 2.5% (v/v) AEMP-TM7279 7.1 mg/ml in    carrier A or B or C

(ii) Concentration Complex 4.2 A B ANMP-MG7274 2.5% (v/v) 3.0% (v/v)ANMP-TM7279 2.5% (v/v) 3.0% (v/v) AEMP-TM7279 10.5 mg/ml 18.5 mg/ml incarrier in carrier A or B or C A or B or C

-   V. Complex 5 comprises ASMP and ANMP in a suitable carrier, in    example

Concentration Complex 5 A B ANMP-MG7274 1.75% (v/v) 3% (v/v)AN{overscore (M)}P-TM7279 1.75% (v/v) 3% (v/v) ASMP-CA9656 15.6 mg/ml15.6 mg/ml in carrier in carrier A or B or C A or B or C

Example 7

The safety of different antigenic preparations was tested in vaccinationexperiments in animal model systems (white mice, guinea pigs, andhorses).

Antigenic fractions were prepared as described in Examples 1 to 3 and 6from Trichophyton mentagrophytes DSM No. 7279, Microsporum gypseum DSMNo. 7274, or Candida albicans DSM No. 9656.

The following clinical observations concerning the condition of thevaccinated animals were made daily up to five days after eachvaccination:

-   1. Common Condition    -   appetite    -   influence on locomotion-   2. Local reaction    -   oedema and inflammation at the injection site    -   changes of the temperature at the injection site    -   development of pain at the injection site    -   necessity to treat the injection site with medicaments-   I. Antigenic preparations were injected one or two times with an    interval of 10 days intra abdominally into white mice and intra    abdominally and sub cutaneously into guinea pigs. The antigenic    preparations, their concentrations and the results are shown in    Tables 5 and 6 (A and B). The subcutaneous or intra abdominal    injection of the fungal antigens as single or complex preparations    mostly had no adverse effect on the general condition of the animals    and a local reaction at the injection site was not observed.-   II. Complex preparations of the fungal antigens as described in    Example 6 (Complexes 4.1, 4.2, and 5) were each once injected intra    muscular into the same horse at different locations (left and right    side of the neck and in one of the chest muscles. Three different    horses were vaccinated: (i) one pregnant mare, (ii) one foal, age    7–8 months, and (iii) one stallion, age: 6 years. The antigenic    preparations, their concentrations and the results are shown in    Table 7.

The intra muscular injection of the fungal antigens as complexpreparation had no influence on the general condition of the horses anda local reaction at the injection site was not observed. These studiesdemonstrate the excellent safety of the antigenic preparations of thepresent invention.

Example 8

The influence of different antigenic preparations on the condition ofskin and hairy coat was studied in white mice.

The antigenic preparations were prepared as described in Examples 1 to 3and 6 from Trichophyton mentagrophytes DSM No. 7279, Microsporum gypseumDSM No. 7274, or Candida albicans DSM No. 9656.

The antigenic preparations were injected two times in an interval of 10days intra abdominally into white mice. Observation of the condition ofskin and hairy coat continued for five days. The antigenic preparations,their concentrations and the results are shown in Table 8. Injections ofthe antigenic preparations improved the condition of skin and hairy coatof white mice, as compared to control animal afflicted with dermatitis.

Example 9

The efficacy of three different antigenic preparations was studied byvaccination of Icelandic horses afflicted with Summer Eczema in aplacebo controlled trial.

The antigenic preparations were prepared as described in Examples 1 to 3and 6 from T. mentagrophytes DSM No. 7279, M. gypseum DSM No. 7274 and Calbicans DSM No. 9656. A volume of 1 ml of Carrier A containing theindividual antigenic preparations was injected three times intramuscularly. The interval between each injection was five days.Injections were administered alternately in the right and left side ofthe chest muscle. The antigenic preparations, their concentrations andthe results are shown in Tables 9 and 10.

Administration of an antigenic preparation comprising ASMP-MG7274,ASMP-TM7279, and ASMP-CA9656 resulted in the complete cure of allvaccinated horses (3) four weeks after the third injection. The horsesof the control group (injection of Carrier A without antigens) did notshow any signs of recovery.

Example 10

The safety of three different antigenic preparations was studied byvaccination of Icelandic horses afflicted with Summer Eczema in aplacebo controlled trial.

The antigenic preparations were prepared as described in Examples 1 to 3and 6 from T. mentagrophytes DSM No. 7279, M. gypseum DSM No. 7274 andC. albicans DSM No. 9656. A volume of 1 ml of Carrier A containing theindividual antigenic preparations was injected three times intramuscularly. The interval between each injection was five days.Injections were administered alternately in the right and left side ofthe chest muscles. Animals were observed for side effects during a timespan of three days after each injection. The antigenic preparations,their concentrations and the results are shown in Table 11. General sideeffects like fever or loss of appetite were not observed. Only one ofthe antigenic preparations induced swelling at the injection-side. Thisminor side effect was observed in only one horse. No signs of pain wereobserved.

Example 11

The antiallergic efficacy of single fractions ASMP-TM7279, ASMP-MG7274and ASMP-CA9656 as well as of complex 1 comprising ASMP-TM7279,ASMP-MG7274 and ASMP-CA9656 has been studied in a laboratory animalmodel.

Single fractions have been prepared according to example 1. Complex 1was prepared according to examples 1 and 6.

CF-1 mice have been sensitzed following the modell and instructions ofthe Mouse Ear Swelling Test (Gad S C, Dimm B K, Dobbs D W, Reilly C,Walsh R D: Development and Validation of an Alternative DermalSensitization Test: The Mouse Ear Swelling Test (MEST). Toxicology andApplied Pharmacology 84, 93–114, 1986. This is a well known, validatetand OECD accepted test to examine allergic substances. To prove theefficacy of the complex or its single fractions for its anti allergicpotency in a laboratory animal ear swelling which is caused by theallergene should be prevented. A placebo controled blind study with miceand two different allergens had been conducted:

The MEST was performed with CF-1 mice, which are most sensitive forallergenes. 6–10 week old CF-1 mice, have been prepared by shaving theabdominal skin, injecting 0,05 ml of Freund's Adjuvans and applying 100μl of the allergene 1-chloro-2,4-dinitrocholorbenzene (DNCB) in onetrial and mite allergene in another trial topically to the shavedabdominal skin from day 0 to 4. Seven days later 20 μl of the allergenehad been applied topically to the test ear, the dissolving solution tothe control ear. 24 and 48 h later the ear thickness have been measured.The same procedure has been carried out with the control group, whichhas been treated with placebo instead of the complex respectively thefractions of the complex.

Administration of single fractions ASMP-TM7279, ASMP-MG7274 andASMP-CA9656 as well as of complex 1 comprising ASMP-TM7279, ASMP-MG7274and ASMP-CA9656 resulted in 90% reduced ear swelling after sensitizationwith mite allergene and 87,5% reduced ear swelling after sensitizationwith DNCB 48 h following rechallenge in comparison to the controlgroups.

Example 12

The efficacy of a complex preparaton, comprising antigenic preparationsASMP-MG7274 and ASMP-CA9656, prepared as described in Example 1, wasdemonstrated by vaccination of an Icelandic horse afflicted with SummerEczema.

Intradermal injections of a volume of 0.4 ml of Carrier A containing 0.2mg of MG and 0.2 mg of CA for three times, with an interval of five daysbetween each injection, resulted in the cure of the vaccinated horsethree weeks after the final injection, as evidenced by significantdecrease of the clinical symptoms. No side effects have been observed.

Example 13

The efficacy of an antigenic preparation prepared as described inExample 1 (ASMP) from Microsporum gypseum DSM No. 7274 was demonstratedby vaccination of an 41 year old man suffering from an eczema withinflammation, itching and erosions on the skin between the 4th and 5thtoe.

A volume of 0.1 ml of Carrier A containing 0.4 mg of ASMP-MG7274 wasinjected intradermally, once only. The skin turned to normal 4–5 daysafter treatment.

Itching already had disappeared 24 h after injection. No severe sideeffects have been observed.

Example 14

The efficacy of an antigenic preparation prepared as described inExample 1 (ASMP) from Candida albicans DSM No. 9656 in the treatment ofneurodermitis was demonstrated.

ASMP-CA9656 was mixed into a cream, using “Kamill Hand und Nagelcreme”purchased from Procter & Gamble, to a final concentration of 60 mgASMP-CA9656/ml cream. The preparation was applied topically to a 3 yearold girl suffering from neurodermitis with yellow crusts on the skinnear both ears. The cream was applied topically to the defect part ofskin once per day for 30 days. After this treatment the skin returned tonormal. Side effects have not been observed.

Example 15

The efficacy of an antigenic preparation prepared as described inExample 1 (ASMP) from Microsporum gypseum DSM No. 7274 in the treatmentof eczema was demonstrated.

ASMP-MG7274 was mixed into a cream, using “Kamill Hand und Nagelcreme”purchased from Procter & Gamble, to a final concentration of 60 mgASMP-CA9656/ml cream. A 30 year old man suffering of an eczema withinflammation, erosions and itching on the ring finger was treated bytopical application of the aflicted parts of the skin once per day for30 days. This resulted in complete cure after treatment. Itching haddisappeared a few days after treatment start. Side effects have not beenobserved.

Example 16

The efficacy of antigenic preparations, prepared as described in Example1 (ASMP) from Microsporum gypseum DSM No. 7274, Trichophytonmentagrophytes DSM No. 7279, and Candida albicans DSM No. 9656, has beentested by vaccination in a 5 year old horse which had not changed thewinter coat till June. A volume of 1 ml of Carrier A containing 15 mg ofeach antigenic preparation ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656(final concentration 45 mg ASMP/ml) was injected three times with aninterval of five days intramuscularly, what resulted in complete changeto regular season coat within 15 days. Side effects have not beenobserved.

Example 17

The efficacy of a complex antigenic preparations prepared as describedin Example 1 (ASMP) from Microsporum gypseum DSM No. 7274, Trichophytonmentagrophytes DSM No. 7279, and Candida albicans DSM No. 9656 for thetreatment of alopecia is demonstrated.

Two 7 year old horses suffering from alopecia one 3–5 and one on 7–10different locations all over the body were treated with a vaccinecontaining 10 mg of each antigenic preparation ASMP-MG7274, ASMP-TM7279and ASMP-CA9656 in 1 ml of carrier A (final concentration 30 mg/ml). Thevaccine was injected three times with an interval of five daysintramuscularly, what resulted in complete restitution of the coat ofboth horses 10 days after the last application. Side effects have notbeen observed.

Example 18

The efficacy of a complex antigenic preparation prepared as described inExample 1 (ASMP) from Microsporum gypseum DSM No. 7274, Trichophytonmentagrophytes DSM No. 7279, and Candida albicans DSM No. 9656 for thetreatment of alopecia in horses is demonstrated.

A 10 year old horse suffering from alopecia on 10–12 different locationsall over the body was treated with a vaccine containing 15 mg of eachantigenic preparation ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656 in 1 mlof carrier A (final concentration 45 mg/ml). The vaccine was injectedthree times with an interval of five days intramuscularly, what resultedin complete restitution of the coat 15 days after the last application.Side effects have not been observed.

Example 19

The efficacy of a complex antigenic preparation prepared as described inExample 1 (ASMP) from Microsporum gypseum DSM No. 7274, Trichophytonmentagrophytes DSM No. 7279, and Candida albicans DSM No. 9656 for thetreatment of alopecia in dogs is demonstrated.

A 3 year old female dog suffering from alopecia on 2–3 differentlocations all over the body was treated with a vaccine containing 10 mgof each antigenic preparation ASMP-MG7274, ASMP-TM7279 and ASMP-CA9656in 1 ml of carrier A (final concentration 30 mg/ml). The vaccine wasinjected three times with an interval of five days intramuscularly, whatresulted in complete restitution of the coat 15 days after the lastapplication. Side effects have not been observed.

Example 20

The efficacy of a complex antigenic preparation prepared as described inExample 1 (ASMP) from Microsporum gypseum DSM No. 7274, Trichophytonmentagrophytes DSM No. 7279, and Candida albicans DSM No. 9656 for thetreatment of alopecia in dogs is demonstrated.

Two male dogs, one 5 years and one 8 years old, suffering from alopeciaon 2–4 different locations all over the body were treated with a vaccinecontaining 15 mg of each antigenic preparation ASMP-MG7274, ASMP-TM7279and ASMP-CA9656 in 1 ml of carrier A (final concentration 45 mg/ml). Thevaccine was injected three times with an interval of five daysintramuscularly, what resulted in complete restitution of the coat 30days after the last application. Side effects have not been observed.

TABLE 2 Influence of the concentration of different antigenic fractionson the growth of keratinocyte cell cultures (HaCat cells in percent,compared to controls not exposed to antigenic fractions (confluent cellmono layer of controls = 100%) concentration of antigenic fraction[mg/ml] antigenic 0.003 0.007 0.01 0.015 0.03 0.1 0.3 0.45 0.6 1.0 1.251.5 1.75 2 fraction area covered by cells in percent compared tocontrols ASMP-MG7274 100 100 100 100 100 75 50 25 25 25 0 0 0 0ASMP-TM7279 100 100 100 100 100 100 75 50 25 0 0 0 0 0 ASMP-CA9656 100100 100 100 100 100 100 100 100 75 50 25 25 0 ANMP-MG7274 100 100 100100 100 100 100 100 100 75 75 50 25 0 ANMP-TM7279 100 100 100 100 100100 100 100 100 75 50 25 25 0 ANMP-CA9656 100 100 100 100 100 100 100100 100 75 75 50 25 0 AEMP-MG7274 100 100 100 100 100 100 75 75 50 25 00 0 0 AEMP-TM7279 100 100 100 100 100 100 100 100 100 75 75 50 25 0AEMP-CA9656 100 100 100 100 100 100 100 100 100 75 50 25 0 0

TABLE 3 Concentration of different antigenic fractions resulting in 50%growth inhibition of keratinocyte cells (HaCaT cells) concentration ofantigenic fraction [mg/ml] strain ASMP ANMP AEMP MG7274 0.3 1.5 0.6TM7279 0.45 1.25 1.5 CA9656 1.25 1.5 1.25

TABLE 4 Influence of different antigenic fractions on cell proliferationof horse lymphocytes concentration horse lymphocyte proliferation in %(controls without of antigenic exposure to antigenic fractions = 100%)fraction ASMP- ASMP- AEMP- AEMP- AEMP- [μg/ml] ASMP-MG7274 TM7279 CA9656MG7274 TM7279 CA9656 500 81.5 36.2 37.8 19.4 7.0 1.08 50 117 106.9 109.876.1 10.2 46.9 5 181 172.8 119.8 129.7 47.3 138.3 0.5 98.6 93.8 134.1147.8 133.3 138.3 0.05 271.3 94.3 181.5 143.5 94.8 149.8 0.005 146.7207.6 144.7 104.7 109.9 146.3

TABLE 5A Reaction of test animals (white mice, body weight 12–14 g)after “first injection” of individual antigenic fractions number ofanimals showing concentration injection number of Impairment antigenic[mg/ml] or volume animals local local increase of local loss of oflethal fraction [ % (v/v)] [ml] vaccinated pain reaction temperatureappetite locomotion reactions ASMP-MG7274 10.5 mg/ml 0.5 10 0 0 0 0 0 0ASMP-TM7279 12.5 mg/ml 0.5 10 0 0 0 0 0 0 ASMP-CA9656 15.5 mg/ml 0.5 9 00 0 0 0 0 ANMP-MG7274 3.3% 0.5 8 0 1 0 0 0 0 ANMP-TM7279 3.3% 0.5 10 0 20 0 0 0 ANMP-CA9656 3.3% 0.5 10 0 1 0 0 0 0 AEMP-MG7274 15.5 mg/ml 0.510 0 1 0 0 0 0 AEMP-TM7279 11.3 mg/ml 0.5 10 0 1 0 0 0 0 AEMP-CA965613.5 mg/ml 0.5 10 0 1 0 0 0 0

TABLE 5B Reaction of test animals after “first injection” of complexpreparations number of animals showing concentration injection number ofImpairment [mg/ml] or volume animals local local increase of local lossof of lethal complex No [ % (v/v)] [ml] vaccinated pain reactiontemperature appetite locomotion reactions white mice (body weight 12–14g) ANMP-MG7274 4.2  2.5% 0.5 10 0 0 0 0 0 0 ANMP-TM7279  2.5%AEMP-TM7279 10.5 mg/ml ANMP-CA9656 4.1  2.5% 0.5 10 0 0 0 0 0 0AEMP-TM7279  7.1 mg/ml ANMP-MG7274 5 1.75% 0.5 10 0 0 0 0 0 0ANMP-TM7279 1.75% ASMP-CA9656 15.6 mg/ml Guinea pigs (body weight150–200 g) ANMP-MG7274 4.2  2.5% 0.5 5 0 0 0 0 0 0 ANMP-TM7279  2.5%AEMP-TM7279 10.5 mg/ml ANMP-CA9656 4.1  2.5% 0.5 5 0 0 0 0 0 0AEMP-TM7279  7.1 mg/ml ANMP-MG7274 5 1.75% 0.5 5 0 0 0 0 0 0 ANMP-TM72791.75% ASMP-CA9656 15.6 mg/ml

TABLE 6A Reaction of test animals (white mice, body weight 12–14 g)after “second injection” of individual antigenic fractions number ofanimals showing concentration injection number of Impairment antigenic[mg/ml] or volume animals local local increase of local loss of oflethal fraction [ % (v/v)] [ml] vaccinated pain reaction temperatureappetite locomotion reactions ASMP-MG7274 10.5 mg/ml 1.0 10 0 0 0 0 0 0ASMP-TM7279 12.5 mg/ml 1.0 10 0 0 0 0 0 0 ASMP-CA9656 15.5 mg/ml 1.0 9 00 0 0 0 0 ANMP-MG7274 3.3% 0.5 8 0 1 0 0 0 0 ANMP-TM7279 3.3% 0.5 10 0 20 0 0 0 ANMP-CA9656 3.3% 1.0 10 0 1 0 0 0 0 AEMP-MG7274 15.5 mg/ml 0.510 0 1 0 0 0 0 AEMP-TM7279 11.3 mg/ml 1.0 10 0 1 0 0 0 0 AEMP-CA965613.5 mg/ml 0.5 10 0 1 0 0 0 0

TABLE 6B Reaction of test animals after “second injection” of complexpreparations number of animals showing concentration injection number ofImpairment [mg/ml] or volume animals local local increase of local lossof of lethal complex No [ % (v/v)] [ml] vaccinated pain reactiontemperature appetite locomotion reactions white mice (body weight 12–14g) ANMP-MG7274 4.2  2.5% 0.5 10 0 0 0 0 0 0 ANMP-TM7279  2.5%AEMP-TM7279 10.5 mg/ml ANMP-CA9656 4.1  2.5% 0.5 10 0 0 0 0 0 0AEMP-TM7279  7.1 mg/ml ANMP-MG7274 5 1.75% 0.5 10 0 0 0 0 0 0ANMP-TM7279 1.75% ASMP-CA9656 15.6 mg/ml Guinea pigs (body weight150–200 g) ANMP-MG7274 4.2  2.5% 0.5 5 0 0 0 0 0 0 ANMP-TM7279  2.5%AEMP-TM7279 10.5 mg/ml ANMP-CA9656 4.1  2.5% 0.5 5 0 0 0 0 0 0AEMP-TM7279  7.1 mg/ml ANMP-MG7274 5 1.75% 0.5 5 0 0 0 0 0 0 ANMP-TM72791.75% ASMP-CA9656 15.6 mg/ml

TABLE 7 Reaction of horses after injection (single injection) of complexpreparations (each horse received complexes No. 4.1, 4.2 and 5 at thesame time in separate injections at separate locations) number of horseswith number of horses with general reactions: concentration injectionnumber of local reactions Impairment [mg/ml]or volume horses oedema/increase of loss of of loss of complex No [ % (v/v)] [ml] vaccinatedpain inflammation temperature appetite locomotion animals ANMP-MG72744.2 3.0% 0.5 3 0 0 0 0 0 0 ANMP-TM7279 3.0% AEMP-TM7279 18.5 mg/mlANMP-CA9656 4.1 3.0% 0.5 0 0 AEMP-TM7279 15.6 ANMP-MG7274 5 3.0% 0.5 0 0ANMP-TM7279 3.0% ASMP-CA9656 15.6

TABLE 8 Condition of skin and hairy coat after injection of complexantigenic preparations in white mice (body weight 12–14 g) number ofnumber of animals animals showing showing the concen- the followingfollowing tration condition condition of the hairy [mg/ml] injection ofthe skin coat after vaccination: or [ml] number of after vaccination:smooth touseling complex No [ % (v/v)] first second animals scalingsmooth and shining and dim ANMP-MG7274 4.2 2.5% 0.5 1.0 2 0 2 2 0ANMP-TM7279 2.5% AEMP-TM7279 10.5 mg ANMP-CA9656 4.1 2.5% 0.5 1.0 3 0 33 0 AEMP-TM7279 7.1 mg ANMP-MG7274 5 1.75% 0.5 1.0 1 0 1 1 0 ANMP-TM72791.75% ASMP-CA9656 15.6 mg not vaccinated — — — — 3 3 0 2 1

TABLE 9 Efficacy of vaccination with different complex preparationsdetermined in Iceland horses afflicted with Summer Eczema injec- fourweeks after tion third vaccination: concentration vol- number of numberof horses [mg/ml] or ume number of horse cured not cured complex No [ %(v/v)] [ml] injections vaccinated from Summer Eczema ASMP-MG7274 1 10mg/ml 1 3 3 3 0 ASMP-TM7279 10 mg/ml ASMP-CA9656 10 mg/ml ANMP-MG7274 21% 1 3 3 0 3 ANMP-TM7279 1% ANMP-CA9656 1% AEMP-MG7274 3 10 mg/ml 1 3 31 2 AEMP-TM7279 10 mg/ml AEMP-CA9656 10 mg/ml

TABLE 10 Efficacy of vaccination with different complex preparations,determined in Iceland horses afflicted with Summer Eczema concentrationfour weeks of individual after third fractions injection: [mg/ml]injection number of number of horses or volume number of horses curedfrom complex No [ % (v/v)] [ml] injections vaccinated itching eczemaASMP-MG7274 1 10 mg/ml 1 3 3 3 3 ASMP-TM7279 10 mg/ml ASMP-CA9656 10mg/ml ANMP-MG7274 2 1% 1 3 3 1 0 ANMP-TM7279 1% ANMP-CA9656 1%AEMP-MG7274 3 10 mg/ml 1 3 3 2 1 AEMP-TM7279 10 mg/ml AEMP-CA9656 10mg/ml no antigen — — 1 3 3 0 0 (Carrier A only) (control)

TABLE 11 Safety of vaccination with different antigenic preparationsdetermined in Iceland horses afflicted with Summer Eczema number ofhorses with number of horses with general side effect concentrationlocal side effects observed observed 1 to 3 days after [mg/ml] injectionnumber number of 1 to 3 days after first to first to third injection orvolume of horses third injection loss of complex No [% (v/v)] [ml]injections vaccinated swelling pain fever appetite ASMP-MG7274 1 10mg/ml 1 3 3 0 0 0 0 ASMP-TM7279 10 mg/ml ASMP-CA9656 10 mg/mlANMP-MG7274 2 1% 1 3 3 0 0 0 0 ANMP-TM7279 1% ANMP-CA9656 1% AEMP-MG72743 10 mg/ml 1 3 3 1 0 0 0 AEMP-TM7279 10 mg/ml AEMP-CA9656 10 mg/ml noantigen — — 1 3 3 0 0 0 0 (Carrier A only) (control)

TABLE 12 NMR-Spectra endstanding antigenic douplet triplet multipletisolated CH2 alcyl-CH aryl-H amino preparations acetate (s) amino acidsamino acids carbohadrates amino acids amino acids acids ASMP MG 7274/1.92 ppm CH3 (d, 6.8 Hz) CH3 (t, 7.1 Hz) 3.2–4.3 ppm 1.7–3.45 ppm 0.95ppm 9-18-1 1.33 ppm 1.18 ppm (FIG. 4) CH3 (d, 7.5 Hz) 4.9–5.4 ppm 1.48ppm CH2(AcB, 16 Hz) 2.7 ppm/2.5 ppm CA 9656/ 1.92 ppm CH3 (d, 7.0 Hz)CH3 (t, 7.0 Hz) 3.4–4.6 ppm 3.28 ppm 0.95 ppm b008 1,33 ppm 1.18 ppm(FIG. 2) CH3 (d, 7.3 Hz) 4.9–5.24 ppm 1.48 ppm TM 7279/ 1.92 ppm CH3 (d,7.1 Hz) CH3 (t, 7.1 Hz) 3.5–4.35 ppm 2.1–3.3 ppm 0.85–0.95 ppm 32-m-1-51.33 ppm 1.18 ppm (FIG. 3) CH3 (d, 7.1 Hz) 5.0–5.25 ppm 1.48 ppm AEMP TM7279/ CH3 (d, 6.5 Hz) 3.2–4.07 ppm 1.6–3.12 ppm p32-5-1 1.33 ppm (FIG.1, CH3 (d, 7.5 Hz) 0.84–1.08 ppm 7.15–7.9 ppm A–C) 1.48 ppm CH (d, 8.2Hz) 4.65 ppm CH (d, 4.0 Hz) 5.23 ppm ppm = part per million s = singulet

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1 to 4;

NMR experiments of ASMP and AEMP fractions as shown in FIGS. 1 to 4 wereperformed according to the following:

Spectra have been obtained in D₂O on a 250 MHZ BRUKER digitalNMR-spectrometer (model DRX 400) with a ¹H-frequency of 400.13 Mc. Sweepwidth is 14.5 ppm, ambient temperature is 300K. Chemical shifts arereferenced by means of the solvent distance.

The standard ¹H-one dimensional spectra have been obtained using theappropriate BRUKER pulse program.

1. A process for the preparation of an antigenic material soluble inaqueous solutions (ASMP) which comprises at least one polysaccharide andat least one glycopeptide, said process consisting of: a) contactingfungal cells, which belong to keratinophilic fungi or keratinophilicyeasts, or parts thereof with an aqueous alkaline solution at about 20°C. to about 150° C., thereby obtaining a preparation; b) separating saidpreparation into solid and liquid phases by centrifugation or bysedimentation, thereby obtaining a first supematant; c) contacting saidfirst supernatant with mineral or organic acid and allowing solid andliquid phases to separate, thereby obtaining a second supematant; and d)precipitating out of said second supematant the ASMP.
 2. The process asclaimed in claim 1, wherein a) the fungal cells or parts thereof arecontacted with KOH or NaOH of about 0.1–5% (w/v), at about 20–150° C.,for up to 30 hours, thereby obtaining the preparation; b) centrifugingsaid preparation, thereby obtaining the first supernatant; c) contactingsaid first supernatant with 0.2–1.5M organic acid or 0.05–1M mineralacid, centrifuging, thereby obtaining a second supernatant; and d)precipitating out of said second supernatant the ASMP by contacting saidsecond supernatant with an organic solvent or a salt.
 3. The process asclaimed in claim 2, wherein said organic solvent or a salt is selectedfrom the group consisting of an alcohol, a lower alkanol and ammoniumsulphate.
 4. The process as claimed in claim 2, further comprising: e)dissolving said ASMP in an aqueous solution.
 5. The process according toclaim 1, wherein said keratinophilic fungus is selected from a group oftwo fungi genera consisting of Trichophyton and Microsporum; and saidyeast is in the genus Candida.
 6. The process according to claim 1,wherein said keratinophilic fungus is selected from a group consistingof Trichophyton equinum, Trichophyton mentagrophytes, Trichophytonsarkisovii, Trichophyton verrucosum, Microsporum canis, Microsporumgypseum, and Candida albicans.
 7. The process according to claim 1,wherein said keratinophilic fungus is selected from a group consistingof Trichophyton equinum DSM No. 7276, Trichophyton mentagrophytes DSMNo. 7279, Trichophyton sarkisovii DSM No. 7278, Trichophyton verrucosumDSM No. 7277, Microsporum canis DSM No. 7281, Microsporum canis var.obesum DSM No. 7280, Microsporum canis var. distortum DSM No. 7275,Microsporum gypseum DSM No. 7274, and Candida albicans DSM No.
 9565. 8.An antigenic material comprising the ASMP material obtained by a processas claimed in claim 1, and at least one material selected from the groupconsisting of antigenic material not soluble in aqueous solutions (AMNP)and antigenic exogenous material (AEMP).
 9. The antigenic material asclaimed in claim 8, wherein the antigenic material is a combination ofASMP from strain T. mentagrophytes DSM No. 7279, ASMP from strain M.gypseum DSM No. 7274, and ASMP from strain C. albicans DSM No.
 9656. 10.The antigenic material as claimed in claim 8, wherein the antigenicmaterial is a combination of ASMP from strain M. gypseum DSM No. 7274and ASMP from strain C. albicans DSM No.
 9656. 11. A pharmaceuticalcomposition comprising at least one antigenic material as claimed inclaim 8 in a pharmaceutically acceptable carrier.
 12. The composition asclaimed in claim 11, wherein the antigenic material is in a solution forinjection.
 13. A vaccine comprising at least one antigenic material asclaimed in claim
 8. 14. The vaccine as claimed in claim 13, furthercomprising a pharmaceutically acceptable carrier.
 15. An antigenicmaterial comprising at least one of the materials selected from a groupconsisting of ASMP, ANMP, and AEMP; wherein said material is obtained bya process as claimed in claim
 7. 16. A pharmaceutical compositioncomprising at least one antigenic material obtained by a process asclaimed in claim 5 in a pharmaceutically acceptable carrier.
 17. Apharmaceutical composition comprising at least one antigenic materialobtained by a process as claimed in claim 6 in a pharmaceuticallyacceptable carrier.
 18. A pharmaceutical composition comprising at leastone antigenic material as claimed in claim 15 in a pharmaceuticallyacceptable carrier.
 19. A vaccine comprising at least one antigenicmaterial obtained by a process as claimed in claim
 5. 20. A vaccinecomprising at least one antigenic material obtained by a process asclaimed in claim
 6. 21. A vaccine comprising at least one antigenicmaterial as claimed in claim
 15. 22. A vaccine that comprises antigenicmaterial obtained by a process as claimed in claim 1.